Untreated HIV-1+ individuals frequently suffer from HIV associated neurocognitive disorders (HAND) including HIV associated dementia (HAD). In the era of HAART, milder impairments persist and may increase with long-term therapy, while severe neurocognitive disorders including HAD occur in subjects who fail therapy. Brain tissue is colonized early in infection. However, proviral DNA is hard to detect or undetectable in brain during the asymptomatic phase. Regardless, long-lived viral reservoirs become established in macrophages, microglia and astrocytes, each of which will need to be eradicated if effective cures are realized. A current hypothesis is that the brain is reseeded late in disease by infected monocytes that increasingly migrate through the blood brain barrier. While this model is attractive, supporting data is limited and it is also possible that uninfected monocytes entering the brain late on differentiate into macrophages and amplify highly macrophage-tropic variants long established there. Many reports also describe astrocyte infection, however, some remain unconvinced. In addition, astrocytes do not express CD4 and mechanisms of infection are unclear while the virus present in this reservoir remains uncharacterized. We will investigate the viral reservoirs inside and outside the brain that carry HIV-1 variants related to highly mac-tropi variants in the brains of AIDS patients with HAD or normal neurology. We propose 3 aims: Aim 1: To investigate reservoirs that seed HIV-1 into the brain We will investigate whether bone marrow and blood monocytes reseed HIV-1 into the brain in late disease or whether the influx of uninfected monocytes late on acts to expand virus already present in the brain. Aim 2: To characterize of HIV-1 reservoirs associated with glial precursor cells and astrocytes in comparison with macrophage:microglia We will use magnetic bead enrichment and sorting of specific brain cells, single genome PCR and phylogenetic analysis to investigate HIV-1 in macrophage/microglia, glial precursor and astrocyte populations. Aim 3: To track brain env motifs and related variants in tissue reservoirs by deep sequencing. We will use deep sequencing approaches to measure the tissue distribution and prevalence of HIV-1 variants in brain and their relatives outside focusing on brain portals. In summary, we will elucidate the tissues and cell types inside and outside the brain that carry variants related to the predominant quasispecies in brain. Our data will provide insights into the portals that seed HIV-1 into and out of the brain and will provide a comprehensive view of the tissue distribution and prevalence of quasispecies that establish the major reservoirs of HIV in brain tissue. Our study represents basic research on the HIV-1 reservoirs inside and outside the brain and will have relevance for the development of new eradication approaches to eliminate HIV-1 from brain tissue.